Breynia cernua: Chemical Profiling of Volatile Compounds in the Stem Extract and Its Antioxidant, Antibacterial, Antiplasmodial and Anticancer Activity In Vitro and In Silico

Breynia cernua has been used as an alternative medicine for wounds, smallpox, cervical cancer, and breast cancer. This plant is a potential source of new plant-derived drugs to cure numerous diseases for its multiple therapeutic functions. An in vitro study revealed that the methanol extract of B. cernua (stem) exhibits antioxidant activity according to DPPH and SOD methods, with IC50 values of 33 and 8.13 ppm, respectively. The extract also exerts antibacterial activity against Staphylococcus aureus with minimum bactericidal concentration of 1875 ppm. Further analysis revealed that the extract with a concentration of 1–2 ppm protects erythrocytes from the ring formation stage of Plasmodium falciparum, while the extract with a concentration of 1600 ppm induced apoptosis in the MCF-7 breast cancer cell line. GC–MS analysis showed 45 bioactive compounds consisting of cyclic, alkyl halide, organosulfur, and organoarsenic compounds. Virtual screening via a blind docking approach was conducted to analyze the binding affinity of each metabolite against various target proteins. The results unveiled that two compounds, namely, N-[β-hydroxy-β-[4-[1-adamantyl-6,8-dichloro]quinolyl]ethyl]piperidine and 1,3-phenylene, bis(3-phenylpropenoate), demonstrated the best binding score toward four tested proteins with a binding affinity varying from −8.3 to −10.8 kcal/mol. Site-specific docking analysis showed that the two compounds showed similar binding energy with native ligands. This finding indicated that the two phenolic compounds could be novel antioxidant, antibacterial, antiplasmodial, and anticancer drugs. A thorough analysis by monitoring drug likeness and pharmacokinetics revealed that almost all the identified compounds can be considered as drugs, and they have good solubility, oral bioavailability, and synthetic accessibility. Altogether, the in vitro and in silico analysis suggested that the extract of B. cernua (stem) contains various compounds that might be correlated with its bioactivities.


Introduction
From a historical point of view, medicinal plants have been proven to have therapeutic properties to cure various diseases, and they still represent an important source of novel drugs today [1,2]. More than 50% of drugs in modern therapeutics are represented by plant-derived drugs, either in their natural or in their derivative form [3,4]. For example, successful plant-derived drugs are aspirin from Filipendula ulmaria (L.) Maxim and artemisinin from Artemisia annua L. [5,6]. Indonesia is a country with high plant biodiversity and has very diverse plant resources (approximately 30,000-40,000 plant species), 20% of which are either wild or cultivated medicinal plants [7].
On the other hand, metabolite profiling and the exploration of medicinal plants in Indonesia are still rarely conducted. For those reasons, the metabolite profile of uninvestigated plants in Indonesia is interesting to explore since they are a promising source of new plant-derived drugs. Therefore, this research aimed to unveil the metabolite component of one herbaceous plant as a reservoir of a particular bioactive compound that can potentially be used as a drug in the future.
Various Breynia species have been traditionally applied as medicine for numerous diseases [8]. One such species, Breynia cernua (local name: Katuk Hutan), has been used in Amuntai, Hulu Sungai Utara, and Kalimantan Selatan as herbal medicine for smallpox and wounds [9], as well as in Papua as a traditional medicine for cervical and breast cancer [10]. B. cernua has the following anatomical characteristics: semilunar vascular bundle, paracyitic and anomocytic stomata, irregular shape of the lower epidermis with a wavy anticlinal wall, polygonal shape of the upper epidermis with a straight anticlinal wall, single unicellular trichome on the lower epidermis, one-layer upper epidermis with a regularly shaped cell, one-layer lower epidermis with a papillated cell, one-layer elongated palisade tissue, loose arrangement of the spongy tissue, druse calcium oxalate on vascular bundle parenchyma, and hypodermis in the area near leaf bone [11]. It was found that the ethanol extract of B. cernua has cytotoxic activity according to the brine shrimp lethality test and MTT assay against MCF-7 [10]. The ethyl acetate or butanol extract of B. cernua (stem hardwood) and methanol extract of B. cernua (stem bark) exhibited broad-spectrum antibacterial activities against Micrococcus luteus, Micrococcus roseus, Bacillus coagulans, Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Citrobacter freundii, Lactobacillus casei, Staphylococcus albus, Staphylococcus aureus, Staphylococcus epidermidis, Agrobacterium tumefaciens, Streptococcus faecalis, Streptococcus pneumoniae, Klebsiella pneumoniae, Eschericia coli, Proteus vulgaris, Proteus mirabili, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Enterobacter aerogenes, Salmonella Typhi, Salmonella Typhimurium, and Serratia marcescens. Furthermore, the butanol or methanol extract of B. cernua (root bark) demonstrated antifungal activities against Aspergillus niger, Aspergillus versicolour, Aspergillus vitis, Candida albican, Candida tropicalis, Cladosporium cladosporides, Penicillium notatum, Trichophyton mentagrophyte, Tricophyton rubrum, and Tricophyton tonsurans [12]. The ethanol extract of B. cernua also was found to have antiparkinsonism activity in vitro and in vivo [13]. Considering its multiple in vivo and in vitro bioactivity, B. cernua is a promising source of novel plant-derived drugs. Therefore, identification of its chemical composition and activity prediction of each metabolite represent interesting research pathways.
Traditional medicinal plant research has gradually risen worldwide in recent years, owing to the natural sources and variety of such plants, which allow them to complement current pharmaceutical treatments [14,15]. The bioactivities of medicinal plants are correlated with the phytochemicals contained in them such as alkaloids, flavonoids, saponins, terpenoids, tannins, and quinonens [16]. Metabolite profiling through GC-MS using the silyl derivatization method has been widely applied to identify the composition of plant extracts [17,18]. In addition, bioinformatics tools aid drug discovery by using all primary data gathered from in vivo and in vitro studies in a short time and at a lower cost. In silico molecular docking is one of the most effective ways to find novel ligands for proteins with known structures, thus playing an essential role in structure-based drug development [19]. A preliminary study (GC-MS) showed that the stem extract of B. cernua contained more compounds than the leaf extract. Moreover, the stem extract exhibited better antioxidant and bactericidal activity than the leaf extract (manuscript in preparation). Therefore, in this report, we performed metabolite profiling, characterized the in vitro antioxidant, antibacterial, antiplasmodial, and anticancer activity, and conducted virtual drug screening through molecular docking, drug likeness, and pharmacokinetic analyses of the methanol extract of B. cernua (stem).

Plant Material and Preparation of the Extracts
Breynia cernua was collected from a coal mining reclamation area at Site Kusan-Girimulya of PT Borneo Indobara. The plant material's taxonomic identification was validated by reference books and research articles [20]. To obtain the extract, 200 g of B. cernua stems were cut and oven-dried (50 • C, 48 h) before being macerated in 100 mL of methanol (24 h, room temperature). The solvent was removed by oven-drying (70 • C, 4 h), and the extract was solubilized using DMSO (0.5% v/v).

Gas Chromatography/Mass Spectrometry (GC-MS) Method
GC-MS was used to identify compounds in the methanol extract of B. cernua (stem). Sample derivatization was conducted using the trimethyl silyl derivatization (TMS) method. Briefly, 20 µL of extract was dried using an oven (80 • C, 20 min). Immediately after, the sample was solubilized using 80 µL of methoxyamine hydrochloride solution in pyridine (2 mg/100 mL), mixed by vortexing for 1 min, and incubated for 90 min at 30 • C. Next, the solubilized sample was combined with 80 µL of MSTFA and incubated for 30 min at 37 • C prior to GC-MS analysis [21]. GC-MS analyses of B. cernua (stem) extracts were carried out using an Agilent 5977B GC/MS (made in USA). Pure helium gas was employed as the carrier gas (1 mL/min), and the injector temperature was set at 270 • C. The oven temperature program was set as follows: gradual increase from 70 • C to 200 • C (10 • C/min), then to 310 • C (10 • C/min), before holding at 310 • C for 5 min. The mass spectrometer was set to electron ionization mode at 70 eV with an electron multiplier voltage of 1859 V. The retention index of compounds was recorded using Mass Hunter GC/MS Acquisition 10.0.368 software. The phytochemical content determination was conducted by comparing the results to authentic compound spectral databases stored in the National Institute of Standards and Technology (NIST) library.

Antioxidant Activity Test
The antioxidant activity test was conducted using DPPH and SOD methods. The assay was repeated with solvent (DMSO 0.5% v/v) as a negative control.

DPPH Method
First, 4 × 10 −4 M DPPH solution was prepared and placed into a vial, closed tightly and wrapped using aluminium foil. From the stock solution (1000 ppm), the sample concentration was varied from 0.5 to 42 ppm and placed in test tubes. After that, 1 mL of DPPH solution was added to each test tube and allowed to sit for 30 min, followed by absorbance measurements (λ517 nm). The following equation was used to calculate the ability to scavenge DPPH free radicals (inhibition): %h = (Ab -As)/Ab × 100%, where %h is the percentage inhibition (free-radical inhibition), Ab is the absorbance blank, and As is the sample absorbance. The value of 50% free-radical inhibition concentration (IC 50 ) was calculated using the regression equation y = ax + b.

Method for Superoxide Dismutase (SOD) Activity Assay
From the stock solution (1000 ppm), the sample concentration was varied from 5 to 20 ppm and placed in test tubes. The SOD activity was measured using a xanthinexanthine oxidase system capable of generating superoxide radicals. At λ550 nm, the degree of suppression of nitro blue tetrazolium reduction by O 2 was measured. The amount of enzyme required to inhibit nitro blue tetrazolium degradation by 50% in 1 min was measured as U/mg protein [22]. The following equation was used to calculate the percentage inhibition: %h = (Ab -As)/Ab × 100%, where %h is the percentage inhibition (free-radical inhibition), Ab is the absorbance blank, and As is the sample absorbance. The value of 50% free-radical inhibition concentration (IC 50 ) was calculated using the regression equation y = ax + b.

Minimum Inhibitory Concentration against Staphylococcus aureus
The broth microdilution method was used to determine the minimum inhibitory concentration (MIC) according to CLSI [23]. The extracts were serially diluted twice in a microtiter plate with Mueller-Hinton broth. Each plate received a bacterial inoculum with a final concentration of 5 × 10 5 CFU/mL and was incubated at 37 • C for 24 h. The MIC was defined as the lowest extract's concentration that inhibited bacterial growth. The minimum bactericidal concentration (MBC) was considered as the lowest extract concentration resulting in no bacterial growth. Treatment with solvent (DMSO 0.5% v/v) was also used as a negative control.

Antiplasmodial Assay
P. falciparum strain FCR-3/Gambia was purchased from ATCC (30932 TM ). The cells were grown in Roswell Park Memorial Institute's medium (RPMI 1640 R8578) supplemented with inactivated blood type O serum in a 9:1 ratio. Serum was extracted from whole blood and centrifuged for 10 min at 1600 rpm. The serum was then inactivated by heating it at 56 • C for 30 min. P. falciparum cells were cultivated in a complete medium in red blood cells (RBCs) at 37 • C and 5% CO 2 . The medium was regularly replaced twice a week. After 1 week of maintenance (all RBCs were lysed by parasites), the cells were ready to be used for the next experiment. The red blood cells (RBCs) were obtained from blood type O, which was centrifuged at 1600 rpm for 10 min. The pellet of RBC was washed in phosphate-buffered saline solution and resuspended with complete medium in a ratio of 1:1. For treatment, infected RBCs were prepared by adding 1 × 10 6 RBCs to complete medium and 10 µL of a P. falciparum cell suspension in a 50 mL Falcon tube. Extracts of B. cernua at various concentrations (600 ppm, 300 ppm, 150 ppm, 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, 2 ppm, or 1 ppm) were administered to the same Falcon tube. Treatment with solvent (DMSO 0.5% v/v) was also used as a negative control. Suspended cells were grown for 2 × 24 h on six-well plates at 37 • C and 5% CO 2 . The positive control consisted of RBCs and P. falciparum, while the negative control consisted of RBCs. Then, cells were harvested to calculate the number of intact RBCs using a hemocytometer, and the infected RBCs were observed using Giemsa staining. Giemsa staining was applied by placing 10 µL of cell suspension on a slide, fixing it with methanol, and incubating it with Giemsa solution for 10 min. The presence of P. falciparum in RBCs was determined using a light microscope.

Cytotoxic Activity
The MCF-7 human breast cancer cell line was purchased from the ATCC (HTB-22 TM ) (transcriptome sequence data were deposited in the ArrayExpress database https://www. ebi.ac.uk/biostudies/arrayexpress/ (accessed on 21 May 2022) by Chiang et al. with accession number E-MTAB-609) [24]. The cells were grown in Eagle's minimum essential medium (EMEM) supplemented with 1% penicillin/streptomycin and 10% heat-inactivated fetal bovine serum in a 96-well plate (37 • C, 5% CO 2 , 20 h). Then, the cells were treated with various concentrations of B. cernua stem extract (200, 400, 800, and 1600 ppm) for 3, 6, 8, and 24 h. Treatment with solvent (DMSO 0.5% v/v) was also used as a negative control. The cell viability was estimated by measuring absorbance at λ570 nm using a Quant ELISA plate reader (Agilent, CA, USA). Trypan blue staining was used to measure the level of cell death. The morphology of cells was observed using inverted microscopy (Agilent).

Drug Likeness and Pharmakokinetic Analysis
Drug likeness and pharmacokinetic analyses of molecules were analyzed using the SwissADME server (http://www.swissadme.ch/)(accessed on 8 May 2022). The drug likeness analysis was conducted according to the criteria of Lipinski, Ghose, Veber, Egan, and Muegge.

Antioxidant and Antibacterial Test Results
The extract exhibited remarkable antioxidant activity as measured using the DPPH (2,2-diphenyl-1-pikrilhidrazil) and SOD (superoxide dismutase) methods, with IC 50 values of 33 and 8.13 ppm, respectively. Numerous phenolic compounds can result in antioxidant activity since the phenolic and flavonoid groups commonly act as antioxidants, negating the oxidative damage generated by free radicals at the molecular level [76]. In addition, the extract also possessed bactericidal activity against pathogenic bacterium S. aureus at a concentration of 1875 ppm. This finding is in line with a prior study on B. cernua extract, which exhibited broad-spectrum antimicrobial activity against bacteria, including S. aureus [12].

Antiplasmodial Test Result
An antimalarial test was conducted on Plasmodium falciparum FBR3. The test parameters were the number of intact erythrocytes and the parasitemia level of the treatment using both extracts at various concentrations with an incubation period of 2 × 24 h, compared to the negative control. The results demonstrated that the methanol extract was active against P. falciparum. Both methanol extract treatments (1 and 2 ppm) show good activity. The number of erythrocytes decreased on P. falciparum-infected erythrocytes; however, the 1-2 ppm extract treatment protected cells from lysis (Figures 1 and 2). At 10 ppm, the extract seemingly did not have a protective effect since the number of intact erythrocytes was similar to the control (+). Further analysis showed that the methanol extract might play a role in protecting erythrocytes from ring formation stage of P. falciparum infection (Figure 3).

Anticancer Test Result
Previous research stated that n-hexane fraction of B. cernua had the highest cytotoxic toward MCF-7 breast cancer cells compared to ethyl acetate and water fractions, with an IC 50 value of 165.65 ppm. On the basis of these data, a test was conducted on the methanol extract of B. cernua using the MCF-7 cell line. The test was conducted at various concentrations to determine the extract's activity in killing cancer cells. The result showed that the extract started killing cancer cells within 3 h of treatment at concentrations of 1600 ppm and 800 ppm. Concentrations under 800 ppm did not show any effects on the cells. (Figure 4). Further morphology analysis showed that B. cernua (1600 ppm, 3 h) induced cell shrinkage and apoptotic body formation, which are hallmarks of apoptosis ( Figure 5).

Anticancer Test Result
Previous research stated that n-hexane fraction of B. cernua had the highest cytotoxic toward MCF-7 breast cancer cells compared to ethyl acetate and water fractions, with an IC50 value of 165.65 ppm. On the basis of these data, a test was conducted on the methanol extract of B. cernua using the MCF-7 cell line. The test was conducted at various concentrations to determine the extract's activity in killing cancer cells. The result showed that the extract started killing cancer cells within 3 h of treatment at concentrations of 1600 ppm and 800 ppm. Concentrations under 800 ppm did not show any effects on the cells. (Figure 4). Further morphology analysis showed that B. cernua (1600 ppm, 3 h) induced cell shrinkage and apoptotic body formation, which are hallmarks of apoptosis ( Figure 5).

Anticancer Test Result
Previous research stated that n-hexane fraction of B. cernua had the highest cytotoxic toward MCF-7 breast cancer cells compared to ethyl acetate and water fractions, with an IC50 value of 165.65 ppm. On the basis of these data, a test was conducted on the methanol extract of B. cernua using the MCF-7 cell line. The test was conducted at various concentrations to determine the extract's activity in killing cancer cells. The result showed that the extract started killing cancer cells within 3 h of treatment at concentrations of 1600 ppm and 800 ppm. Concentrations under 800 ppm did not show any effects on the cells. (Figure 4). Further morphology analysis showed that B. cernua (1600 ppm, 3 h) induced cell shrinkage and apoptotic body formation, which are hallmarks of apoptosis ( Figure 5).

Virtual Screening with Blind Docking Approach
Virtual screening was used to estimate each compound's affinity to selected target proteins: 3VSL as an antibacterial protein model, 3EUB as an antioxidant protein model, 3QS1 as an antiplasmodial protein model, and 3GD4 as an anticancer protein model. Blind docking performed using Pyrx software revealed that 44 of 45 compounds were successfully docked into the target proteins. However, the docking analysis could not be performed using arsenous acid, tris(trimethylsilyl) because the arsenic atom could not be recognized by AutoDock Vina software ( Table 2).

Virtual Screening with Blind Docking Approach
Virtual screening was used to estimate each compound's affinity to selected target proteins: 3VSL as an antibacterial protein model, 3EUB as an antioxidant protein model, 3QS1 as an antiplasmodial protein model, and 3GD4 as an anticancer protein model. Blind docking performed using Pyrx software revealed that 44 of 45 compounds were successfully docked into the target proteins. However, the docking analysis could not be performed using arsenous acid, tris(trimethylsilyl) because the arsenic atom could not be recognized by AutoDock Vina software (Table 2).    B. cernua is known as an alternative medicine with antiviral properties toward smallpox [9] and anticancer properties toward breast and cervical cancer [10]. In addition, its methanol extract also exerts broad-spectrum antibacterial activity [12], as well as antioxidant, antiplasmodial, and anticancer properties (this study). Computational docking tools are used by researchers all around the world to uncover and evaluate the binding affinity of compounds that fit a protein's binding site [19]. From the above in vitro and in vivo evidence, four protein models were chosen as molecular docking targets, as they were previously crystallized and/or used for in silico studies to investigate the binding affinity of particular compounds: 3EUB as an antioxidant target protein [77], 3VSL as an antibacterial target protein [78], 3QS1 as an antiplasmodial target protein [79], and 3GD4 as an anticancer target protein [80]. According to the virtual screening results (Table 2), N-[β-hydroxy-β-[4-[1-adamantyl-6,8-dichloro]quinolyl]ethyl]piperidine and 1,3-phenylene, bis(3-phenylpropenoate) exhibited the best binding affinity toward all protein models. Hence, the detailed interactions between the two phenolic compounds and the target proteins were further investigated.

Site-Specific Docking on Binding Site of Native Ligands
Site-specific docking was performed to compare the binding energy of N-[β-hydroxyβ- [4-[1-adamantyl-6,8-dichloro]quinolyl]ethyl]piperidine and 1,3-phenylene, bis(3-phenylpropenoate) with the native ligands of each protein. Table 3 reveals that the two compounds exhibited almost similar binding energy to the native ligands of each protein, except for the binding energy of N-[β-hydroxy-β- [4-[1-adamantyl-6,8-dichloro]quinolyl]ethyl]piperidine on MTE binding site of 3EUB, which was much lower than that of the native ligand. Figures S7-S12 visualize the binding interactions between the native ligands and the isolated compounds and each target protein. The result demonstrates that the two compounds have potential application as novel drugs for the inhibition of antibacterial, antioxidant, antiplasmodial, and anticancer protein targets in a competitive manner. Nevertheless, an experimental investigation is needed to validate these findings. Therefore, the next step will be to obtain these compounds naturally or synthetically.

Drug Likeness and Pharmacokinetic Analyses
The qualification of each identified compound as an oral drug was conducted according to the criteria of Lipinski, Ghose, Veber, Egan, and Muegge (Table 4). Lipinski's rule of five is as follows: molecular weight < 500 Da, <5 hydrogen bond donors, <10 hydrogen bond acceptors, and log P < 5. Ghose utilizes four parameters to qualify a molecule as drug: 160 Da ≤ molecular weight ≤ 480 Da, −0.4 ≤ log P ≤ 5.6, 40 ≤ omlar refractivity ≤ 130, and 20 ≤ total atom number ≤ 70. Veber bases drug likeness on two parameters: topological polar surface area (TPSA) < 140 A 2 and number of rotatable bonds <10. Egan considers three criteria: TPSA <132 A 2 and −1 ≤ log P ≤6. Muegge establishes drug likeness as fol-lows: 200 Da ≤ molecular weight ≤ 400 Da, −2 ≤ log P ≤ 5, TPSA ≤ 150 A 2 , <5 hydrogen bond donors, <10 hydrogen bond acceptors, <15 rotatable bonds, <7 rings, and at least four carbon atoms and one heteroatom. Solubility was measured using the ESOL model, where a log S value ≤−6 indicates a poorly soluble compound, while a log S value ≤−10 indicates an insoluble compound. Additionally, a compound with 0.55 ≤ bioavailability score ≤ 0.85 and 0.25 ≤ Csp3 (degree of flexibility) score ≤1 is qualified as orally bioavailable [82]. Compounds with no more than one violation are promising drug candidates. According to Lipinski's criteria, all of the identified molecules in the methanol extract of B. cernua (stem) can be considered oral drug candidates. According to Ghose's criteria, almost all molecules can be considered drug candidates except for compounds 1 and 10. The filter process using Veber and Egan criteria showed that all molecules qualified as drug candidates. On the basis of Muegge's criteria, only compounds 4,5,8,10,14,21,22,23,27,35,36,37,39, and 41 qualified as drug candidates. The bioavailability and Csp3 score revealed that all compounds were orally bioavailable except compound 22.  The pharmacokinetic parameters of each compound (Table 5) were predicted in terms of gastrointestinal absorption (GI), blood-brain barrier (BBB) permeability, and skin permeability (log Kp). A more negative log Kp indicates lower skin permeability of the molecule. Another factor underlying pharmacokinetics is the interaction between the molecule and particular proteins such as permeability glycoprotein (P-gp) and cytochromes P450 (CYPs). P-gp is a key factor in active efflux toward the biological membrane, while CYPs are proteins that play a role in drug biotransformation. The synthetic accessibility score ranges from 1 (very easy) to 10 (very difficult) [83]. Table 5 shows that half of the compounds had high GI absorption, whereas the other half had low GI absorption. To solve this challenge, scientists have been using particular strategies such as encapsulating drugs in lipid formulations [84]. The pharmacokinetic analysis also revealed that half of the compounds exhibited low penetration into the BBB and half of the compounds exhibited high penetration into the BBB. Generally, a low penetration of drugs into the BBB is required to minimize the side-effects on the central nervous system (CNS). On the other hand, drugs with high permeability across the BBB are needed to cure cancer patients with brain metastases [85]. The prediction of the interactions with pharmacokinetic proteins revealed that almost all compounds had a low interaction with CYP enzymes, thus indicating minimal drug-drug interactions. Moreover, all compounds had easy to moderate synthetic accessibility. In view of this thorough analysis, these compounds can be good drug leads of natural origin. Further studies on the purification or synthesis of these can be conducted to reveal the mechanisms underlying their antioxidant, antibacterial, antiplasmodial, and anticancer activities.

Conclusions
As a country with huge biodiversity, Indonesia has abundant plant resources to be used as herbal medicine or as metabolite reservoirs for drug discovery. Amongst them, B. cernua is a potential medicinal plant to be explored since it has been traditionally used to treat various diseases. We provided in vitro evidence that the methanol extract of B. cernua (stem) exhibits antioxidant, antibacterial, antiplasmodial, and anticancer activities. In addition, the plant has been traditionally demonstrated to have antiviral activity. Further analysis revealed that the extract induced apoptosis in the MCF-7 breast cancer cell line and protected erythrocytes from the ring formation stage of P. falciparum. Then, to identify important phytoconstituents, the extract's metabolites were profiled using GC-MS analysis, which revealed 45 compounds that could contribute to the plant's therapeutic functions. Bioinformatics was used for drug discovery to save time and chemical reagents. Therefore, blind docking was performed to identify the binding affinity of the metabolites toward target proteins, which revealed that N-[β-hydroxy-β-[4-[1-adamantyl-6,8dichloro]quinolyl]ethyl]piperidine and 1,3-phenylene, bis(3-phenylpropenoate) exhibited the best results. Further analysis showed that the two molecules were predicted to have an inhibition effect toward antioxidant, antiplasmodial, and anticancer protein models in a competitive manner. Meanwhile, the two molecules were predicted to have an inhibitory effect toward the antibacterial protein model in a noncompetitive manner. Site-specific docking analysis showed that the two compounds exhibited similar binding energy to the native ligands. Interestingly, their bioactivities have not yet been documented. The drug likeness and pharmacokinetic analyses revealed that almost all identified compounds can be considered promising drugs, showing good oral bioavailability, high absorption, and good synthetic availability. Our results clearly indicate that the in silico studies were supported by in vitro studies.